(a) Field of the Invention
The invention relates to compounds and the use of novel peptide and peptidomimetic antagonists of a G protein coupled receptor, in treatments focused on regulating the fluid filtration in the kidney in case of acute renal failure, end stage renal disease, glomerulonephritis and other nephropathies, on decreasing resorption and bone mineral loss as in osteoporosis and dental diseases and additionally, closure of ductus arteriosus (DA) in premature infants or fetal animals.
(b) Description of Prior Art
Prostaglandins are derived from the oxygenation of arachidonic acid by prostaglandin (PG) synthases. Prostaglandins mediate a wide variety of physiological actions, such as vasomotricity, sleep/wake cycle, intestinal secretion, lipolysis, glomerular filtration, mast cell degranulation, neurotransmission, platelet aggregation, leuteolysis, myometrial contraction and labor, inflammation and arthritis, patent ductus arteriosus, cell growth and differentiation. Prostanoids mediate their actions through binding to distinct receptors which belong to the super family of rhodopsin-like seven transmembrane helical receptors. These receptors are coupled to heterotrimeric G-proteins comprising of α,β and γ subunits which, upon activation, elicit alterations in cell calcium, initiate phosphoinositide hydrolysis or promotion or repression of cyclic adenosine monophosphate synthesis.
Of the five pharmacologically-distinct prostanoid receptors for PGE2, PGI2, PGD2, PGF2α and TxA2, four subtypes of PGE2 receptor are described (Ichikawa, et al. 1996). These are EP1, EP2, EP3 which has several splice variants and EP4. Cloned human EP4 (also known as prostaglandin E2 receptor subtype EP4) is a 488 amino acid glycoprotein, linked to Gαs and involved in stimulation of adenylate cyclase and cAMP synthesis (Abramovitz, M. et al., U.S. Pat. Nos. 5,759,789 and 5,605,814). EP4 receptor is expressed at a high level in intestine, but at much lower levels in lung, kidney, thymus, uterus and brain (Bastien, Y. et al. 1994 J. Biol. Chem. 269 (16):11873-77). EP4 is expressed in ductus arteriosus (Bhattacharya, M. et al. 1999. Circulation. 100:1751-56). Paradoxically, EP4 knock-out mice die after birth due to insufficient closure of ductus arteriosus (Nguyen, M. et al. 1997. Nature. 390:78-81; Segi, E. et al., 1998). Hence the mechanism of ductal patency and the role of EP4 remain elusive.
PGE2 is abundantly produced in kidney and is involved in the regulation of renal microcirculation, salt and water transport, renin release (Breyer, M. D. et al. 1998. Kidney Int. 54 (Suppl. 67): S88-94). All EP receptors have been shown to be regionally distributed in the kidney structures (Morath, R. et al. 1999. J. Am. Soc. Nephrol. 10: 1851-60) and are associated with specific functions. All studies on the distribution of EP receptors in kidney showed that EP4 receptor is uniquely expressed in glomeruli (Breyer, M. D. et al. 1998. Am. J. Physiol. 270: F912-918; Morath, R., 1999), however the presence of this receptor in other structures of the nephron, such as collecting duct (Breyer, M. D., et al. 1998), the media of renal arteries and vasa recta (Morath, R. et al. 1999) is variously reported. EP4 transcripts were also found in juxtaglomerular granule cells and is consistent with PGE2-induced cAMP synthesis in these cells; hence EP4 may also play a role in renin secretion. Glomerular prostaglandins are thought to affect filtration (Schlondoff, D. et al. 1986. Kidney Int. 29: 108-19) and renin release. PGE2 increases cAMP levels in isolated glomeruli (Freidlander, G. et al., 1983. Mol. Cell. Endocrinol. 30: 201-214). It is suggested that EP4 receptor coupled to cAMP synthesis may regulate glomerular filtration (Sugimoto, Y. et al., 1994), though a direct demonstration of its role is lacking. Most importantly, there is no data in the literature on whether stimulation or antagonism of EP4 receptor would result in increased glomerular filtration or how improving glomerular filtration could have therapeutic benefits in acute renal disease end stage renal disease, glomerulonephritis and diabetic nephropathy.
Bones undergo continuous remodeling in which bone formation is carried out by osteoblasts and bone resorption is carried out by osteoclasts. These processes are controlled by several humoral factors such as parathyroid hormone, estradiol, vitamin D, cytokines, growth factors and prostaglandins. It is known that osteoclast induction by interleukin-1 (IL-1) is inhibited by aspirin-like drugs (Tai, H., et al., 1997. Endocrinology. 138: 2372-2379). PGE2 analogs with EP4 receptor agonistic activity promoted osteoclast formation in cocultures of mouse osteoblasts and bone marrow cells and similar experiments using cells from EP4-knockout mice resulted in reduced osteoclast formation, suggesting a role for EP4 receptor in osteoclastogenesis in mice (cited in Narumiya, S. et al. 1999. Physiol. Rev. 79: 1193-1226). Hence it is expected that EP4 antagonists would have therapeutic benefits in medical conditions such as osteoporosis, dental diseases and other diseases where bone loss is integral part of the disease process. Lack of selective antagonists to EP4 receptor hindered progress in this area of research; it is therefore one of the objects of the present invention to overcome one or more of these deficiencies in the art.
It would be desirable to be provided with a prostaglandin E2 receptor subtype EP4 receptor antagonist capable of inhibiting at least one functional consequence of said receptor activity.
It would be desirable to be provided with a method of use for such an antagonist in a suitable pharmaceutical formulation with the purpose of improving glomerular filtration and/or urine output of a patient while treating end stage renal disease glomerulonephritis, diabetic nephropathy or acute renal failure, or closing ductus arteriosus (DA) in a premature infant patient, or preventing further bone loss in osteoporosis, dental disease, and other medical conditions where bone loss is a problem.
In order to further the search for small molecule antagonists of EP4 receptor using highthroughput screening approaches, it is desirable to provide a bioassay or a kit in which appropriately labelled antagonist of EP4 receptor of the present invention could be used as a ligand.